Modulation of nNOS ser852 phosphorylation and translocation by PKA/PP1 pathway in endothelial cells

Nitric Oxide. 2018 Jan 30;72:52-58. doi: 10.1016/j.niox.2017.11.007. Epub 2017 Nov 26.

Abstract

Neuronal nitric oxide synthase (nNOS) is now considered an important player in vascular function. It has a protective role in atherosclerosis and hypertension. However, despite its importance, little is known about the mechanisms that regulate its activity in vascular cells. Here we explore the mechanisms by which nNOS is activated in endothelium. We evaluated aorta relaxation response and phosphorylation of nNOS during protein phosphatases 1 and 2 (PP1 and PP2) inhibition, in eNOS silenced mice. PP1 translocation and interaction between the nuclear inhibitor of PP1 (NIPP1) and PP1 was evaluated in endothelial EA.hy926 cells. We demonstrate here that acetylcholine (Ach)-induced relaxation is completely abolished by nNOS inhibition in eNOS silenced mice aorta which also decreased NO and H2O2 concentrations. ACh induced dephosphorylation of nNOSser852 in aorta after 20 min stimulation. Endothelial cells also showed a decrease in nNOSser852 phosphorylation during 20 min of ACh stimulation. PP2 inhibition had no effect on Ach-induced nNOSSer852 dephosphorylation in endothelial cells and did not modify Ach-induced vasodilation in aorta from eNOS silenced mice. Non-selective PP1/PP2 inhibition prevented nNOSSer852 dephosphorylation in endothelial cells and prevented Ach-induced vasodilation in eNOS silenced mice. ACh induced time-dependent PP1 and NIPP1 dissociation and PP1 translocation to cytoplasm. Protein kinase A (PKA) inhibition abolished PP1 translocation and further nNOSser852 dephosphorylation. In addition, 8-Br-cAMP reduced NIPP1/PP1 interaction, stimulated PP1 translocation and nNOSser852 dephosphorylation. Moreover, PKA Inhibition led to a decreased nNOS translocation to perinuclear region. Taken together, our results elucidate a mechanism whereby PP1 is activated by a cAMP/PKA-dependent pathway, leading to dephosphorylation of nNOSser852 and subsequent NO and possible H2O2 production resulting in endothelium-dependent vascular relaxation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholine / pharmacology
  • Animals
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Endothelium, Vascular / cytology
  • Humans
  • Hydrogen Peroxide / metabolism
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Male
  • Mice, Inbred C57BL
  • Nitric Oxide Synthase Type I / metabolism*
  • Nitric Oxide Synthase Type III / genetics
  • Nitric Oxide Synthase Type III / metabolism
  • Phosphorylation
  • Protein Phosphatase 1 / metabolism*
  • Protein Transport
  • Serine / metabolism
  • Vasodilation / physiology

Substances

  • Intracellular Signaling Peptides and Proteins
  • protein phosphatase inhibitor-1
  • Serine
  • Hydrogen Peroxide
  • Nitric Oxide Synthase Type I
  • Nitric Oxide Synthase Type III
  • Nos1 protein, mouse
  • Nos3 protein, mouse
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Phosphatase 1
  • Acetylcholine